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Mass spectrometry imaging (MSI) enables the spatial distributions of molecules possessing different mass‐to‐charge ratios to be mapped within complex environments revealing regional changes at the molecular level. Even at high mass resolving power, however, these images often reflect the summed distribution of multiple isomeric molecules, each potentially possessing a unique distribution coinciding with distinct biological function(s) and metabolic origin. Herein, this chemical ambiguity is addressed through an innovative combination of ozone‐induced dissociation reactions with MSI, enabling the differential imaging of isomeric lipid molecules directly from biological tissues. For the first time, we demonstrate both double bond‐ and  sn ‐positional isomeric lipids exhibit distinct spatial locations within tissue. This MSI approach enables researchers to unravel local lipid molecular complexity based on both exact elemental composition and isomeric structure directly from tissues.

Mass Spectrometry Imaging with Isomeric Resolution Enabled by Ozone‐Induced Dissociation